The present invention is in the field of diagnostics and relates more in particular to a method for the determination of biologically active forms of proteolytic enzymes, such as thrombin, in blood and other fluids.
In the Western world, arterial and venous thrombosis and atherosclerosis together are currently responsible for well over 50% of all mortality and serious morbidity. In a few decades this will be the case world-wide [Murray, C. J. and A. D. Lopez, Science (1996) 274:740-743]. Thrombosis is caused by an overactivity of the haemostatic mechanism that is responsible for the arrest of bleeding from a wound. This haemostatic-thrombotic system (HTS) is a complex interaction between vessel wall, blood cells, especially blood platelets, and plasma proteins. See, e.g., Hemker H. C. Thrombin generation, an essential step in haemostasis and thrombosis. In: Haemostasis and thrombosis, A. L. Bloom et al., eds., Churchill Livingstone, Edinburgh, (1994) 477-490.
In blood there are at least three series of proenzyme-enzyme cascades of great biological importance: the blood coagulation system, the fibrinolytic system and the complement system. In each of these systems proteolytic proenzymes are activated and subsequently inhibited. In general it is important that the concentration of an activated enzyme in such a cascade can be measured in order to assess the function of the system. In the following a typical description of the clotting system will be given with an emphasis on its most important enzyme, thrombin. It is to be noted, however, that the present invention also relates to other clotting enzymes and enzymes of the fibrinolytic and complement pathway.
The mechanism of thrombin generation can be outlined in some detail as follows. In the plasmatic coagulation system factor Xa is formed by the action of the tissue factor-factor VIIa complex (TF-VIIe). Factor Xa binds to tissue factor pathway inhibitor (TFPI) and the TFPI-Xa complex inhibits the TF-VIIa complex. Thrombin activates factors V, VIII and XI and so accelerates its own generation, but it also binds to thrombomodulin and so starts the protein C mechanism that breaks down factors V and VIII, thus, indirectly, inhibiting further thrombin generation. An important fraction (≈30%) of all thrombin formed in clotting plasma is bound to the fibrin clot. Clot-bound thrombin does retain its thrombotic properties, it can clot fibrinogen, activate factors V, VIII and XI as well as platelets [Bxc3xa9guin, S. and R. Kumar, Thromb. Haemost. (1997) 78:590-594; Kumar, R., S. Bxc3xa9guin, and H. C. Hernker, Thromb. Haemost. (1994) 72:713-721, and (1995) 74:962-968]. It is not inhibited by antithrombin.
Thrombin action causes receptors in the platelet membrane to bind fibrinogen, which causes platelet aggregation. Platelet activation also leads to the exposure of procoagulant phospholipids in a Von Willebrand factor dependent ii reaction [Bxc3xa9guin S., R. Kumar, I. Keularts, U. Seligsohn, B. C. Coller and H. C. Hemker, Fibrin-Dependent Platelet Procoagulant Activity Requires GPlb Receptors and Von Willebrand Factor, Blood (1999) 93:564-570; Bxc3xa9guin, S. and R. Kumar, supra (1997)]. These phospholipids are required for the proper activation of factor X and prothrombin. Recently, the picture has been complicated by the discovery that fibrin, previously thought to be the inert endproduct of coagulation, plays an active role itself. It binds and activates Von Willebrand factor, which activates platelets and provokes the exposure of procoagulant phospholipids via an alternative pathway.
The cooperation between platelets and the coagulation system, including fibrin, is central to the haemostatic-thrombotic system. The mechanism shows an abundance of positive and negative, often nested, feedback loops. Underactivity causes bleeding, overactivity causes thrombosis. Thrombosis manifests itself as coronary infarction, stroke, pulmonary embolism and a large number of less frequent diseases.
In order to assess the function of such a system, also for diagnostic purposes and for the safe use of antithrombotic drugs, a probe is needed for the functional status of the haemostatic-thrombotic system. An important function test of the HTS is the thrombin generation curve (TGC). Carried out in platelet-poor plasma, it gives information about the function of the plasmatic clotting system. In platelet-rich plasma it measures also the function of the platelets.
According to the prior art the TGC can be measured by subsampling or continuously. In the ancient subsampling method [Biggs, R. and R. G. Macfarlane, Human Blood Coagulation and its Disorders. 1953, Oxford: Blackwell; Quick, A. J., Haemonhagic Diseases. 1957, Philadelphia: Lea and Febiger], samples are taken from a clotting mixture, and the concentration of thrombin is measured in each sample.
Active thrombin survives in plasma for only a limited period of time (the half life time is 16-17 s). This is due to circulating antithrombins. Most thrombin (64%) is inactivated by antithrombin (AT), a plasma-protein of 57 kD, 23% by xcex12-macroglobulin (xcex12M), a 725 kD plasma-protein, and 13% by various other agents [Hemker, H. C., G. M. Willems, and S. Bxc3xa9guin, A computer assisted method to obtain the prothrombin activation velocity in whole plasma independent of thrombin decay processes, Thromb. Haemost. (1986) 56:9-17]. The xcex12-macroglobulin-thrombin complex (xcex12M-IIa) has the peculiarity that it is inactive towards all macromolecular substrates, but retains its activity against small molecular weight (artificial) substrates. xcex12-Macroglobulin is a glycoprotein of Mr 725,000, which is present in plasma in a concentration of 2500 mg/L or 3.5 xcexcM. It is a tetramer of identical subunits of 185 kD. The inactivation reaction of an active proteinase or activated clotting factor with xcex12M is a three step process: 1xc2x0. Formation of a loose complex, 2xc2x0. Hydrolysis of a target peptide in xcex12M, causing 3xc2x0, a rapid conformational change which physically entraps the enzyme molecule within the xcex12M molecule. [See for a review: Travis, J. and G. S. Salvesen, Human plasma proteinase inhibitors. Ann. Rev. Biochem. (1983) 52:655-709].
The group of Hemker then developed a continuous method in which thrombin-catalysed product formation from suitable substrates is monitored directly [Hemker, H. C., et al., Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential. Thromb. Haemost. (1993) 70:617-624]. The kinetic constants of the substrate are such that, at the concentration used, the rate of product formation is proportional to the amount of enzyme present (thrombin or xcex12M-IIa). The time-course of enzyme activity in the sample can be estimated as the first derivative of the product-time curve.
A drawback of this method is that part of the thrombin in plasma binds to xcex12-macroglobulin. xcex12-Macroglobulin is the most abundant non-specific protease inhibitor of blood plasma. It quenches the biological activity of proteases (activated clotting factors, activated fibrinolytic enzymes and activated complement factors) without occupying their active centre, so that there is a residual activity on the usual oligopeptide signal-substrates.
This xcex12-macroglobulin-thrombin complex has no known physiological activity but is able to cleave chromogenic substrates. Thus, the product formation observed is the result of the combined activities of free thrombin and xcex12-macroglobulin-bound thrombin. The relevant data, i.e., the amount of product formed by free thrombin only, can be extracted from the experimental product formation by a mathematical operation. However, the operation has to be carried out on the whole course of the product generation curve. The product formation therefore needs to be monitored continuously. Although the principle of this continuous method is applicable to all substrates that give a product which can be detected in time, e.g., by fluorescent, electrochemical or NMR signals, its application is currently restricted to the use of chromogenic substrates in an optically clear medium, i.e., defibrinated platelet poor plasma (PPP).
Therefore, there is still a need for an efficient method to measure the amount of (active forms of proteolytic enzymes, such as thrombin, in blood and other fluids, which obviates the drawbacks of the prior art. The present invention provides such a method.
In accordance with the present invention, a process is provided for the assessment of an active proteolytic enzyme in a blood or another biological fluid sample possibly comprising a complex of said proteolytic enzyme and xcex12-macro-globulin, wherein said sample is contacted with a substrate comprising a molecule of sufficient size coupled to a signal-substrate, said signal-substrate comprising a detectable leaving group, wherein said substrate is hydrolysed by said proteolytic enzyme but not by said complex.
The active proteolytic enzyme is preferably selected from the group consisting of thrombin, activated clotting factor, activated fibrinolytic factor, and activated component of the complement system. Of these, thrombin is most preferred.
The molecules of sufficient size are preferably selected from the group consisting of inert protein, preferably ovalbumin, polysaccharide, and synthetic polymer. Preferably, these inert molecules are water soluble and have a size such that they just do not fit into the cavity of the xcex12M molecules. A group of preferred inert molecules has a size of about 40 kD and a solubility of at least 40 mg/ml. Most preferred are inert molecules with a size of about 20 kD and a solubility of at least 50 mg/ml.
These and other aspects of the invention will be further outlined in the description which follows.
FIG. 1 represents theoretical curves of hydrolysis products formed in clotting plasma or bloody by hydrolysis of a small thrombin substrate and a large thrombin substrate. Closed black circles indicate the total hydrolysis of a small thrombin substrate; closed gray circles indicate hydrolysis by temporarily present thrombin of a small thrombin substrate; open circles indicate hydrolysis of a large thrombin substrate; closed triangles indicate hydrolysis by formed xcex12-macroglobulin-thrombin complex of a small thrombin substrate.
FIG. 2 represents the curves of hydrolysis products of substrates S2288 coupled with ovalbumin by hydrolysis with xcex12M-IIa and thrombin, respectively. For each curve, the thick gray overlaid curve represents actually measured curves, whereas the thin black curve beneath the thick gray overlaid curve represents the obtained fits.
FIG. 3 represents the fluorescence patterns of the fluorescent substrate Ala-Arg-AMC coupled with ovalbumin by hydrolysis with free thrombin and thrombin inactivated by xcex12M, respectively. The upper curve represents hydrolysis by human thrombin, in which the open circles are actually measured points; the closed black circles are obtained by fitting. The lower curve represents hydrolysis by human xcex12-macroglobulin-thrombin complex, in which the open triangles are actually measured points; the closed triangles are obtained by fitting.
FIG. 4 shows the results of the determination of the ETP in plasma with SQ68 and OA-Phe-Pip-Lys-pNA. Closed triangles represent hydrolysis of OA-Phe-Pip-Lys-pNA in clotting plasma; closed circles represent hydrolysis of SQ68 in clotting plasma; open circles represent hydrolysis of SQ68 in clotting plasma by formed xcex12-macroglobulin-thrombin complex; open triangles represent hydrolysis of SO68 in clotting plasma by temporarily present thrombin.